Imaging cellular events in real time

Find out how Fang Huang, Jeorg Bewersdorf and colleagues use the sCMOS technology in the ORCA-Flash4.0 camera to achieve video-rate imaging at nanometer scales. Read now.

THE QUESTION

How does the model plant Arabidopsis thaliana maintain its circadian clock?

Almost all known organisms maintain an internal clock to coordinate day/night cycles in metabolism and other activities. Past study indicates that eukaryotic circadian clocks rely on inter-dependent negative feedback loops to regulate genes important to daytime and nighttime states.

In the model plant Arabidopsis thaliana, transcription factors that are abundant in the morning inhibit expression of many evening-phased genes. The evening gene products in turn repress their morning counterparts. All known circadian regulators in Arabidopsis act through similar negative feedback circuits.

THE BARRIERS

The promoter regions in most evening-phased Arabidopsis genes share a common segment, called the evening element (EE). Inclusion of the EE in the promoter of a reporter gene confers evening expression, and mutation in the EE decreases expression levels.

Morning-phased gene products have been shown to bind directly to the EE, inhibiting transcription. However, the converse does not hold. Blockade of EE binding does not lead to overexpression of the affected evening genes. Could evening gene regulation require late-day activation of EE, as well as morning repression?

THE SOLUTION

Hsu, et al,1 combined genomics techniques with bioluminescence imaging to identify the protein REVEILLE 8 (REV8) as the first known activator of EE-regulated genes. Using a Hamamatsu ORCA-II ERG camera, the team quantified circadian changes in luminescence from whole, live seedlings expressing luciferase under the control of a circadian-regulated promoter. Mutations of REV8 and closely related proteins lengthened the circadian cycle, and both delayed and reduced expression of multiple EE-regulated genes. In addition, these mutations reduced REV8’s usual EE binding activity. The results indicate that circadian regulation in Arabidopsis involves a complex network of interactions including both negative and positive feedback loops.

THE POSSIBILITIES

Hsu and colleagues relied on Hamamatsu’s ORCA-II ERG camera to track circadian changes in the expression of a bioluminescent reporter gene in living plants. Consider what’s possible with the enhanced resolution and video-enabling speed of newer CMOS technology. Learn more in Exciting Advances Push the Limits of Visualization.